1. Field of Invention
The field of the currently claimed embodiments of this invention relates to microfluidic systems, and more particularly to a multiplexed, continuous-flow, droplet-based platform for high-throughput genetic detection.
2. Discussion of Related Art
High throughput sample processing is a critical requirement for a large number of industries. Some examples include the agricultural, pharmaceutical and biotechnological industries1. As a result, there is a constant drive for innovation in sample processing techniques to support these industries. One major breakthrough in this domain has been the application of various robotic sample handling techniques to improve the speed of sample processing as well as to reduce the volume of reagents used per reaction. Although the robotic systems have become incredibly fast at sample processing operations, they are typically limited to operating with standard multi-well (96, 384 and 1536 well) plates. As a result, the typical sample volume consumption is on the order of microliters per reaction for such systems1. Recent advances in the microfluidic domain show promise in overcoming this limitation of the robotic systems. Droplet-based microfluidic systems have been shown to be capable of performing biomolecular screening with sample volumes as low as picoliters2-6. However, introducing a large number of samples on a miniature microfluidic device is difficult since it is impractical to have hundreds to thousands of sample inlets to a single microfluidic device. Furthermore, the tubing used for supplying the samples to such a microfluidic device would already consume orders of magnitude more sample than is required for the actual analysis on the microfluidic device. So, there is a need for an efficient way to transport a large number of samples to a microfluidic device. Ideally such a sample transport system would be flexible enough to supply variable number of samples to a microfluidic device without any modifications in the transport system or the device.
The ‘plug-in cartridge’ technique developed by the Whitesides group7 provides an elegant solution to the problem of introducing a large number of reagents on a microfluidic device through a single inlet. Under this approach, a series of sample plugs are loaded into a capillary, with air bubbles present between sample plugs acting as spacers. This capillary is connected to a microfluidic device, for serial delivery of these sample plugs. However, in this approach, the sample plugs are constantly in contact with the capillary inner surface, leading to the problem of cross contamination between plugs7. Another modification of this approach developed by the Ismagilov group8 utilizes an immiscible carrier fluid instead of an air bubble to act as a spacer between sample plugs. The carrier fluid in this approach preferentially wets the inner surface of the capillary, thus preventing direct contact between sample plugs and the capillary surface. As a result, the problem of cross contamination between sample plugs is eliminated. The carrier fluids typically used for generating these sample plug arrays are fluorinated oils, which also reduce the problem of reagents leaking from sample plugs into the carrier fluid due to their low solubility for most reagents8.
Although this approach is promising, the current techniques used under this approach for generating the ‘sample plug cartridges’ have some issues which need to be resolved. The common technique of using a syringe pump for aspirating sample plugs from a sample well9-11 in a multi-well plate can be extremely slow. Another technique of using vacuum for aspirating a sample plug can be much faster7. However, this technique can only provide a maximum driving pressure of 1 atm (˜15 psi). As a result, the driving force may not be sufficient to load large numbers of sample plugs into a capillary due to the increasing fluidic resistance of the capillary with the introduction of sample plugs. Furthermore, both of these techniques require the free end of the capillary to be attached to either a syringe or a vacuum source, thus excluding the possibility of operating this sample loading system in sync with the operations on a downstream microfluidic device. This can be a major setback to throughput as the possibility of conducting assays in continuous flow manner on microfluidic devices, as has been demonstrated earlier12, is precluded.
For example, maintaining global crop production has become increasingly important amidst a growing global population, and together with water scarcity, pollution, decreasing arable land, emergence of new pests, and possible climate change pose significant challenges. Toward this end, genetic marker assisted selection (MAS), wherein the plant breeding process relies on known DNA/RNA variations associated with traits of interest (e.g., productivity and disease resistance), has enormous potential to improve the efficiency and precision of conventional plant breeding. Traditional techniques for performing MAS at the scale required for agricultural applications, which requires detecting a large number of markers for a large number of plant samples, involve robotic sample handlers, require large amounts of precious samples (>microliter), and are extremely expensive and relatively slow. Droplet microfluidic devices are capable of high-throughput screening using a compact and inexpensive setup compared to robotic sample handlers, and more importantly, reducing sample volume consumption to the nanoliter regime per reaction, some three orders of magnitude reduction in sample consumption. However, existing droplet microfluidic devices have yet to achieve highly multiplexed genetic detection assays in a continuous-flow and high-throughput manner, thus rendering them impractical for the intended applications. Our platform according to some embodiments of the current invention can solve this critical problem and open the door for droplet microfluidic devices to a host of genetic detection assays for high-throughput MAS.
Therefore, there remains a need for improved multiplexed, continuous-flow, droplet-based platforms for high-throughput genetic detection.